Virulent Clones of Klebsiella pneumoniae: Identification...

Virulent Clones of Klebsiella pneumoniae: Identificationand Evolutionary Scenario Based on Genomic andPhenotypic CharacterizationSylvain Brisse1,2*, Cindy Fevre1,2, Virginie Passet1,2, Sylvie Issenhuth-Jeanjean2, Régis Tournebize3,4,

Laure Diancourt1,2, Patrick Grimont2

1 Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, France, 2 Institut Pasteur, Biodiversité des Bactéries Pathogènes Emergentes, Paris, France, 3 Institut

Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France, 4 Unité INSERM U786, Institut Pasteur, Paris, France

Abstract

Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen(causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländer’spneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition ofvirulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical tounderstand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K.pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content,biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clonesthat differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1,clone CC23K1 and clone CC82K1, were strongly associated with PLA and respiratory infection, respectively. Second, only one ofthe two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena andrhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniaesubsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association ofvirulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of thecapsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae andCC82K1 indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23K1 remainsmetabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence ofimportant virulent clones associated with specific infections and provide an evolutionary framework for research into the linksbetween clones, virulence and other genomic features in K. pneumoniae.

Citation: Brisse S, Fevre C, Passet V, Issenhuth-Jeanjean S, Tournebize R, et al. (2009) Virulent Clones of Klebsiella pneumoniae: Identification and EvolutionaryScenario Based on Genomic and Phenotypic Characterization. PLoS ONE 4(3): e4982. doi:10.1371/journal.pone.0004982

Editor: Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, France

Received December 31, 2008; Accepted January 31, 2009; Published March 25, 2009

Copyright: � 2009 Brisse et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work received financial support from Institut Pasteur and from a generous gift by the Conny-Maeva Charitable Foundation. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Klebsiella pneumoniae is responsible for a variety of diseases in

humans and animals [1–3]. Most notoriously, K. pneumoniae is a

prominent nosocomial pathogen mainly responsible for urinary

tract, respiratory tract or blood infections [4]. Isolates from

hospitals often display antibiotic resistance phenotypes [5,6], while

resistance isolates and genetic elements may also spread into the

community [7,8]. Nosocomial infections are caused by highly

diverse K. pneumoniae strains that may be considered as opportu-

nistic, rather than true pathogens, since they mostly affect

debilitated patients [4]. In contrast, serious community infections

due to K. pneumoniae can affect previously healthy persons.

Historically, K. pneumoniae was described as the agent of

Friedländer’s pneumonia, a severe form of lobar pneumonia with

a high mortality [9]. K. pneumoniae is still one of the leading causes

of community acquired pneumoniae in some countries [10,11].

Recently, K. pneumoniae pyogenic liver abscess (PLA), sometimes

complicated by endophthalmitis or meningitis, emerged in Taiwan

and other Asian countries, as well as in other continents [12–17].

Rhinoscleroma and atrophic rhinitis (also called ozaena) are two

chronic and potentially severely disturbing diseases of the upper

respiratory tract, associated respectively with K. pneumoniae subsp.rhinoscleromatis and K. pneumoniae subsp. ozaenae [3,18–21]. Other K.pneumoniae infections that are severe but more rarely reportedinclude meningitis, necrotizing fasciitis and prostatic abscess [22–

24]. Finally, granuloma inguinale (donovanosis) [25] is caused by

uncultivated bacteria, which may belong to K. pneumoniae [26,27].

Factors that are implicated in the virulence of K. pneumoniaestrains include the capsular serotype, lipopolysaccharide, iron-

scavenging systems, and fimbrial and non-fimbrial adhesins [3,28–

31]. The abundant polysaccharidic capsule that typically sur-

rounds K. pneumoniae protects against the bactericidal action ofserum and impairs phagocytosis, and may be regarded as the most

important virulence determinant of K. pneumoniae. Among the 77described capsular (K) types of the serotyping scheme, types K1,

K2, K4 and K5 are highly virulent in experimental infection in

mice and are often associated with severe infections in humans and

PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4982

animals [1,32–35]. K1 isolates were frequent among Friedländer’s

peumonia cases [1,36] and are prominent among PLA cases,

especially those with complications. Serotypes K2, K4 and K5 are

frequent causes of metritis in mares and were also associated with

community-acquired pneumonia [1,36]. Isolates causing rhino-

scleroma are always of type K3 [18,27]. Finally, although their

role as a direct cause of ozaena is not fully established, K.pneumoniae subsp. ozaenae isolates from cases of atrophic rhinitis areof serotype K4 or more rarely K5 [18].

In contrast with the extensive knowledge that has been gathered

on the genotype-virulence relationships in the closely related

species Escherichia coli and Salmonella enterica, virulent clones of K.pneumoniae remain virtually undefined [31,37,38]. Critically, it isunknown whether particular diseases are caused by specific clones

or rather, by the expression of particular virulence determinants.

This distinction is important, as virulence factors may be

horizontally transferred among strains and could be weakly

associated with the genomic background that harbor them, with

clear implications for emergence of new pathogens and for

diagnostic purposes. It is currently unknown whether capsular

types characterize specific clones, in which case the K type may be

useful to identify such clones and to predict the presence of other

associated virulence determinants. Alternately, as is the case in e.g.

Streptococcus pneumoniae [39], K types may be distributed acrossmany unrelated clones due to frequent horizontal transfer of the

cps operon, which is responsible for the synthesis of the capsularpolysaccharide. In this case, a more complex picture is to be

expected for the association of capsular types, other virulence

determinants, and strain genomic background. More generally,

the genetic structure of K. pneumoniae remains virtually unexplored[40,41], and the phylogenetic relationships among virulent strains

causing identical or distinct diseases are therefore unknown. In

addition, the relationships between environmental, carriage or

virulent K. pneumoniae isolates are undocumented. As a conse-quence, limited information on how these strains evolved to

become pathogenic is currently available.

Evolution towards increased virulence can be accompanied by

ecological changes that reflect specialization of pathogenic

bacterial clones to their new lifestyle. For example, evolution of

the particular pathogenic pattern of Shigella or Salmonella entericaserotype Typhi has been paralleled by host restriction and

reduction of metabolic capabilities [42–44]. With the exception

of the well-known reduced metabolic capabilities of K. p. subsp.

rhinoscleromatis and K. p. subsp. ozaenae [27], it is not known whetherthe virulent strains of K. pneumoniae belong to ecologicallyspecialized pathogenic clones.

The purposes of this study were (i) To determine the population

genetic structure of K. pneumoniae, with a particular emphasis on thedefinition of virulent clones and their distinctness from other

strains; (ii) To determine the extent of horizontal transfer of

capsular synthesis (cps) operons among clones; and (iii) Tocharacterize the virulent clones with respect to capsular type,

other known virulence factors, experimental virulence to mice, and

metabolic properties.

Results

1. Restricted levels of genetic diversity and recombiningpopulation structure

Alignment of the seven genes sequences from 235 isolates

showed no insertion/deletion (indel) in six genes. In gene tonB, oneinsertion of two codons (isolate SB3336) and three deletion events

(one of four codons, and two of two codons) were observed.

Excluding these four indels, 129 (4.3%) of the 3,012 nucleotides

positions were polymorphic, four of them corresponding to tri-

allelic single nucleotide polymorphisms (SNPs), thus implying a

total of 133 mutations. The maximal level of nucleotide divergence

among alleles ranged from 0.37% (gapA) to 1.74% (phoE), while thediversity index p (the average number of nucleotide differences persite between any two sequences chosen randomly from the study

sample) ranged from 0.14% (for gapA) to 1.0% (for tonB) (Table 1).Synonymous substitutions were 12 times more frequent than non-

synonymous substitutions. Despite this high degree of sequence

conservation, a total of 117 haplotypes or sequence types (STs)

were distinguished.

Visual inspection of the repartition of polymorphic sites across

the phylogeny of the concatenated sequence suggested that many

polymorphisms have been shuffled by genetic exchange. The

strong network structure obtained after split decomposition

analysis (Figure 1B) confirmed the high level of incompatibilityamong sites, indicative of a pervasive history of intra- and/or

intergenic recombination. Recombination was detected by LDhat

with statistical significance in the two most polymorphic genes,

tonB (r/m ratio, 22.3; p = 0.02) and phoE (r/m ratio, 18.1;p = 0.0084), indicative of intragenic recombination in these genes.

Frequent polymorphisms were too scarce (no more than 1 or 2

Table 1. Nucleotide polymorphism among 235 Klebsiella pneumoniae isolates.

Gene SizeNo. (%) ofpolymorphic sites

No. ofsynonymous sites

No. of non-synonymoussites Ks Ka Ka/Ks p

gapA 450 13 (2.9) 13 0 0.00563 0.000 0.000 0.00142

infB 318 17 (5.3) 15 2 0.01381 0.00007 0.0051 0.00309

mdh 477 21 (4.4) 16 6 0.00697 0.00055 0.079 0.00219

pgi 432 20 (4.6) 19 1 0.0052 0.00043 0.083 0.00157

phoE 420 25 (6.0) 20 5 0.02842 0.00055 0.019 0.00705

rpoB 501 14 (2.8) 11 3 0.00288 0.00136 0.47 0.00174

tonB 414 21 (5.1) 13 8 0.02739 0.00415 0.15 0.01005

concatenate 3,012 129 (4.29) 103 26 0.01192 0.00099 0.083 0.0037

gnd (a) 360 136 (37.8) 142 11 0.208 0.0043 0.021 0.055

(a) Only 177 strains were sequenced.Ks: No. of synonymous changes per synonymous site. Ka: No. of non-synonymous changes per non-synonymous site.p: nucleotide diversity.doi:10.1371/journal.pone.0004982.t001

Klebsiella pneumoniae Clones


polymorphisms above the 0.1 frequency level) in the remaining

five genes to test for recombination. Evidence of homologous

recombination was also provided by the observation of multiple

nucleotide substitutions between STs differing by a single allele

(single locus variants, or SLVs). First, ST16 and ST60 differed

only by six nucleotides at gene tonB (alleles tonB-4 and tonB-8).

Second, ST65 and ST243 were identical except for five SNPs

between their alleles tonB-13 and tonB-25. For these two cases,

import of a recombining segment, rather than independent

mutations in the mismatched gene, seems compelling. In total,

eight allelic mismatches (24%) between SLV pairs involved more

than one SNP and are likely to result from genetic exchange. Of

note, single nucleotide changes may also have been introduced by

homologous recombination, given the very high sequence

relatedness among most alleles. In conclusion, recombination

appears frequent among housekeeping genes in K. pneumoniae.

2. Identification of virulent clones of K. pneumoniaeAs expected given frequent recombination and low levels of

sequence divergence, sequence-based phylogenetic analysis using

PhyML [45] revealed a bushy tree (data not shown) with no

conspicuous internal structure and only few strongly supported

nodes. ClonalFrame [46] failed to estimate the population

parameters, probably due to insufficient polymorphism. The splits

decomposition network (Figure 1B) revealed no internalphylogenetic structure, with few obvious haplotype associations.

In order to reveal relationships among closely related haplotypes

with an approach that is less sensitive to recombination, we used

the minimum spanning tree (MStree) method based upon allelic

profiles. When allowing only one allelic mismatch to assign isolates

to a given clonal complex (CC), 12 CCs were disclosed (the same

groups were identified using eBURST [47]), while the remaining

isolates were distributed into 72 singletons (Figure 1A).

Figure 1. Clonal diversity and relationships among 235 Klebsiella pneumoniae isolates. A. Minimum spanning tree (MStree) analysis ofmultilocus sequence typing (MLST) data for 235 K. pneumoniae isolates, representing 117 sequence types (STs). Isolates of capsular serotypes K1 to K5are colored according to serotype. Each circle corresponds to a sequence type (ST); ST number is given inside each circle. Grey zones surround STsthat belong to the same clonal complex (CC), which is named according to the central ST (the likely founder of the CC). CC65-K2 is delimited by thered triangle (see text). The lines between STs indicate inferred phylogenetic relationships and are represented as bold, plain, discontinuous and lightdiscontinuous depending on the number of allelic mismatches between profiles (1, 2, 3 and 4 or more, respectively); note that discontinuous links areonly indicative, as several alternative links with equal weight may exist. The STs of reference genome strain MGH78578 (ST38) and of the type strainATCC 13883T (ST3) are indicated. B. Split decomposition analysis of concatenated sequences of the seven genes. Numbers at the tip of branches areST numbers. Note the bushy network structure indicative of pervasive homologous recombination. Branches were colored for the clones that arehighlighted on panel A. Note the distribution into unrelated branches of strains with a given capsular (K) type.doi:10.1371/journal.pone.0004982.g001



Remarkably, six CCs corresponded to serotypes K1 to K4, and

these CCs were characterized by their distinctive K type or

pathological origins. Notably, K1 isolates were distributed into two

CCs, CC23K1 and CC82K1 (Figure 1A). CC23K1 was composedof ST23 and ST57, which comprised only K1 isolates, and ST26

and ST163, which included K35 and K61 isolates. Differently,

CC82K1 included only K1 isolates. Remarkably, the four K1

isolates from cases of PLA belonged to CC23K1: three isolates

from Taiwan belonged to ST23 and one isolate from Zaire had

ST57. Other isolates of ST23 were isolated from horses and from

human blood infections (four isolates each; see Table S1).CC82K1 comprised the 15 remaining K1 isolates, none of which

was involved in liver abscess. These isolates included the reference

strain of serotype K1 (A5054, ST82) as well as 11 isolates from

blood or respiratory infection in France between 1976 and 1984.

Likewise, all K2 isolates except CIP 52.204 (ST86) could be

grouped into two distinct and apparently unrelated groups

(Figure 1A). First, CC14K2 comprised STs 14, 78 and 80, allbeing composed of isolates of serotype K2, and also included some

K24 isolates (ST15). ST14 included ESBL-producing isolates from

Curaçao [48] and isolates collected in France and Italy between

1981 and 2002 from urinary, respiratory, blood and cerebro-spinal

fluid (CSF) infections. A second group of K2 isolates was composed

of STs 65, 25 and 243, which together formed one CC, and of

ST66, which differed from ST65 by only two genes (infB and rpoB,

one SNP each). ST65 includes an isolate from a cat infection, one

isolate that caused an epidemics in monkeys at a French zoo [49],

and one human clinical isolate from an anal abscess, while ST25

corresponds to a nosocomial blood isolate (Table S1). ST66corresponds to the reference strain of capsular serotype K2 (B5055)

and the virulent strain CIP 52.145 [34], as well as to isolate 675,

which was used as a vaccine in animals (Table S1). For simplicity,and given the genetic and phenotypic similarity of ST66 strains with

ST65 strains, we will consider ST66 as a member of CC65K2, even

if ST66 does not belong to CC65K2 sensu stricto.

Sets of isolates which belong to a single clonal complex and

share many other common features including K type, virulence

factor content and metabolic profile (see below), likely descend

from a common ancestral strain from which they inherited their

common properties. Therefore, CC23K1, CC82K1, CC14K2 and

CC65K2 may be regarded as four distinct clones. However, their

precise demarcation is rendered difficult, based on MLST data

alone, by the high degree of allele sharing with other K. pneumoniaeSTs, possibly due to their recent evolutionary emergence or to

ongoing allelic exchange with other STs (see discussion).

All K. pneumoniae subsp. rhinoscleromatis isolates were identical at

the seven genes (ST67) except one isolate (839, France, 1982),

which had a single SNP in tonB, resulting in ST68 (Figure 1A).ST67 included CIP 52.210T, the type strain of K. pneumoniae subsp.

rhinoscleromatis, and C5046, the reference strain of capsularserotype K3. In addition, ST67 included nine unrelated clinical

isolates from rhinoscleroma cases, isolated from six countries

between 1954 and 2003. Clearly, subspecies K. pneumoniae subsp.

rhinoscleromatis is highly homogeneous and appears to correspond toa single clone, which we refer to as clone Rhinoscleromatis.

Interestingly, all Rhinoscleromatis isolates differed from all other

strains, including 10 non-rhinoscleroma isolates with serotype K3,

by four or more allelic mismatches. MLST thus clearly demarcates

Rhinoscleromatis isolates from all other K. pneumoniae members.However, it is important to stress that Rhinoscleromatis clearly

belongs to a single genetic pool together with K. pneumoniae(Figure 1B): the average genetic distance between Rhinoscler-omatis and the 115 other STs is 0.54%, while distances among the

115 STs ranged from 0.033% to 0.70%.

All K. pneumoniae subsp. ozaenae isolates formed a single clonalcomplex, CC91oz. ST91 could be inferred as the genetic founder

of this clone (Figure 1A), as all other STs of CC91oz differed fromST91 by a single mismatch, whereas they differed among them by

two mismatches, with the single exception of the pair ST95 and

ST96. ST91 included CIP 52.211T, the type strain of K. pneumoniaesubsp. ozaenae, and the K4 reference strain D5050, as well as thereference strain of type K5 (CIP 52.212 = E5051) and two clinical

isolates. The other STs of CC91oz included K4 clinical isolates

from ozena cases and blood infections, as well as two isolates from

patients with granulomas (ST90 and ST96). Isolates from ozaena

cases were distributed in the three genotypes ST90, ST91 and

ST95. These results indicate that all K. pneumoniae subsp. ozaenaeisolates can be considered as descending from a single ancestor,

forming clone Ozaenae. This clone was well demarcated from the

remaining isolates, as there was only one K. pneumoniae strain(SB169-2, ST97, C-pattern C16a) that had only two allelic

mismatches with clone Ozaenae, while all other K. pneumoniaeisolates, including three non-Ozaenae K5 isolates, had at least four

mismatches with any member of clone Ozaenae. Of note, clone

Ozaenae (6 STs) is more heterogeneous than clone Rhinoscler-

omatis (2 STs) based on the present strain collection, possibly

reflecting a more ancient evolutionary emergence and/or a more

rapid diversification.

3. Capsular types are not strongly associated withgenomic background

No close phylogenetic relatedness was apparent between the

two K1 groups, between the two K2 groups, and between clones

Rhinoscleromatis and Ozaenae with other K. pneumoniae strains ofserotypes K3, K4 or K5 (Figure 1), indicating an independentorigin in distinct genomic backgrounds, rather than a common

ancestral origin. Two evolutionary mechanisms could result in

identical K-types being distributed in unrelated genomic back-

grounds: horizontal transfer of the cps operon, or evolutionaryconvergence. In the latter scenario, similar capsular polysaccha-

ride antigenic structures would be synthesized by phylogenetically

unrelated cps operons that are functionally identical. In order toestimate the phylogenetic relatedness of cps operon structures,isolates were analyzed by PCR-RFLP of the cps operon [50],which can disclose unrelated C-patterns among isolates of a given

K type. The C-pattern of 211 isolates could be established (TableS1; C-patterns available upon request). Clearly, indistinguishableor highly similar C-patterns were observed in unrelated MLST

genotypes (Figure 2): K1 isolates from both CC23K1 and CC82K1

had C-pattern C1a, while K2 isolates of CC14K2 and CC65K2

exhibited the highly similar patterns C2b to C2e (CC14K2) and

C2a (CC65K2). All K3 isolates from clone Rhinoscleromatis and

from the 10 K3 K. pneumoniae isolates in five other STs, had C-pattern C3a or the highly similar patterns C3b to C3d (Figure 2).In particular, C3a was observed in all Rhinoscleromatis isolates as

well as in the unrelated ST3 and ST13. The three variant K3 C-

patterns were observed in ST8 (C3c), ST71 (C3b) and ST153

(C3d). Likewise, the C-pattern C5a was observed in clone

Ozaenae K5 isolates and in K. pneumoniae K5 isolates (ST60,ST61 and ST149), which do not appear phylogenetically related

(Figure 1). Altogether, these data are suggestive of severalindependent historical events of horizontal transfer of the cpsoperon between isolates belonging to distinct clones.

In order to fully demonstrate suspected cases of cps regionhorizontal transfer, we sequenced in 177 relevant isolates, a 360-nt

internal portion of gene gnd, which genomic location is justadjacent of the cps operon [50,51]. A high level of nucleotidepolymorphism was encountered (Table 1), with 136 (38%)



polymorphic sites, with no indel. Thirteen isolates had a gnd

sequence that differed from the 164 other sequences by 6% to

18%. The remaining 164 sequences were still much more variable

than the seven MLST genes. LDhat analysis demonstrated a

strong intra-genic recombination pattern (r/m ratio = 12.5,

p = 0.008), consistent with the highly reticulated structure obtained

using SplitsTree (Figure 2). However, despite frequent intra-genicrecombination, gnd alleles were remarkably similar for isolates of

the same K type (Figure 2), indicating that the associationbetween the gnd gene and the cps operon was not broken down.

Notably, the gnd sequences of K1 isolates from CC23K1 and

CC82K1 were undistinguishable (gnd-12 in both CCs, and gnd-11 in

two strains of ST82, differing at a single SNP from gnd-12).

Similarly, the gnd sequence of Rhinoscleromatis isolates (alleles gnd-

42, gnd-45 and gnd-46) were either identical or highly similar

(Figure 2) to those of K. pneumoniae K3 isolates (gnd-42, gnd-43 andgnd-44), demonstrating their recent common ancestry and their

horizontal transfer into distinct genomic backgrounds. Likewise,

K5 isolates of clone Ozaenae and K5 K. pneumoniae isolates (ST60

and ST61) had identical gnd sequences (gnd-5). Together with

identity of C-patterns, these data demonstrate a common

evolutionary origin of the cps-gnd region in Rhinoscleromatis and

K3 K. pneumoniae isolates, in Ozaenae and K5 K. pneumoniae isolates,

and in both K1 groups. Horizontal gene transfer of entire gnd-cps

region is the most likely explanation for the current distribution of

cps-gnd regions with a unique origin in distinct genomic

backgrounds. The transfer of the gnd-cps region could be inferred

for other K types as well (data not shown).

Different from the above, gnd sequences in K2 isolates of

CC14K2 (gnd-38) and CC65K2 (gnd-16 or gnd-17) were unrelated

Figure 2. Distribution of related capsular operon regions in unrelated clones. The splits tree represents the relationships among gnd allelesas obtained after split decomposition analysis. The distribution of the gnd alleles found in isolates and reference strains of capsular serotypes K1 to K4is indicated by black coloration of sequence types (STs) in the MStree of the corresponding insets. Below the MStree displays are represented the C-pattern of the corresponding isolates. Note that similar or identical gnd and C-patterns are distributed in unrelated STs.doi:10.1371/journal.pone.0004982.g002



(Figure 2). Because the C-pattern from these two CCs are highlyrelated (Figure 2), it is likely that the cps operon was transferredhorizontally without the gnd gene, or that the gnd gene was

replaced in one of the CCs subsequently to a cps-gnd co-transfer

event. However, as allele gnd-38 was also observed in K2 strain

CIP 52.204 (ST86), horizontal transfer of the cps-gnd region has

occurred between this ST and CC14K2.

It is remarkable that the gnd allele in K4 isolates of clone

Ozaenae was the same as observed in most K1 isolates (gnd-12).

This is fully consistent with the observation that K1 and K4 C-

patterns (Figure 2) are highly similar [50] and indicates a closeevolutionary relationships of the cps operons that determine K1

and K4 serotypes.

4. Virulence is associated with clone, rather than with K-type

In order to determine whether the above-identified clones differ

by their virulence potential, the presence of 10 genetic factors

implicated in Klebsiella virulence was assessed by PCR. A total of

102 representative isolates were characterized (Table 2). Whilethe three genes uge, wabG and ureA gave a positive PCR reaction inall isolates, other factors showed unequal repartition across CCs,

resulting in distinctive virulence factor fingerprints of major CCs

(Table 2). Notably, we found sharp differences in virulence genecontent between CC23K1 and CC82K1, as well as between

CC14K2 and CC65K2. Consistent with the location of magA withinthe cps operon of K1 isolates [52], both K1 groups were magA

positive, while magA was not detected in any other isolate.

However, CC23K1 differed from CC82K1 by the presence (100%

vs. 0%, respectively) of genes mrkD coding for the type 3 fimbriae

adhesin, which facilitates adhesion to the basement membranes of

several human tissues [53,54], and allS, coding for the activator of

the allantoin regulon [30]. Interestingly, allS was specific for K1

isolates of CC23K1 members, as it was undetected in CC82K1 and

in non-K1 members of CC23K1 (ST26-K61 and ST163-K35).

CC23K1 was also characterized by a higher prevalence (80%) of

non-fimbrial adhesin CF29K [55], whereas CC82K1 and most

other isolates were negative.

The two K2 groups CC14K2 and CC65K2 also differed by their

virulence gene content. Particularly, isolates of CC14K2, including

its K24 members, were all positive for the iron uptake marker kfu

[31], whereas all CC65K2 isolates were negative. In contrast, rmpA,

the regulator of mucoid phenotype [56], was undetected in CC14

whereas rmpA PCR was positive in 71% of CC65K2 isolates(Table S1).

Clone Rhinoscleromatis was characterized by the complete

absence of kfu and the presence of rmpA. These characteristics alsodistinguished Rhinoscleromatis from other K3 K. pneumoniae

isolates (Table S1). Ozaenae isolates shared the unique property,together with CC82K1, of being negative for mrkD (except for one

isolate).

To determine whether the two K1 clones and the two K2 clones

differ in their virulence, four to nine strains per clone were tested

in mice (Table S1). There was a clear difference in the virulenceof CC14K2 and CC65K2, as no strain (0 out of nine) of the former

was lethal, whereas four out of six CC65 strains killed mice after

five days. The two avirulent CC65 strains were either rmpAnegative (as were all CC14 K2 strains) or negative for fim and mrkD

(Table S1). Likewise, out of seven CC23K1 strains, four K1 strains(ST57 and three of ST23; all rmpA positive) were lethal to mice.

The three avirulent strains were one ST23 K1 strain and the two

non-K1 strains of ST26 and ST163; these three strains lacked

rmpA. In contrast, of the four CC82K1 strains assayed, only one

was slightly virulent to mice, even though rmpA PCR was positive

(Table S1). Hence, virulence to mice of K1 and K2 strainsappeared to differ, depending on the clone they belonged to.

5. Metabolic versatility and evolution of virulent K.pneumoniae clones

In order to determine whether virulent clones of K. pneumoniae

are truly in the process of adapting to a pathogenic lifestyle, rather

than simply representing classical K. pneumoniae strains withparticular combinations of virulence factors, the ability to utilize

99 carbon sources was compared between representative isolates

of the virulent clones and other K. pneumoniae isolates (Figure 3;Table 3). A total of 32 substrates were either utilized by all isolates(n = 16) or by none (n = 16, Figure 3 legend); some of thesesubstrates are useful for identification of the K. pneumoniae species

[27]. However, the remaining substrates showed differences

among K. pneumoniae strains. Interestingly, the pattern of carbon

source utilization correlated closely with MLST-defined clones

(Figure 3; Table 3). Clone Rhinoscleromatis showed a restrictedsubstrate utilization pattern, with the distinctive loss of the ability

to use seven substrates, including D-glucuronate and D-galactur-

Table 2. Virulence gene content of Klebsiella pneumoniae clones (a).

Geneclone Rhinoscleromatis(n = 13)

clone Ozaenae(n = 12)

CC23K1(n = 10) (b)

CC82K1(n = 15)

CC14K2/K24(n = 20) CC65K2 (n = 9)

magA 0 0 100 100 0 0

allS 0 0 100 0 0 0

rmpA 100 41.7 80 86.7 0 77.8

mrkD 100 8.3 100 0 100 88.9

kfu 0 50 100 100 100 0

cf29a 0 8.3 80 0 0 44.4

fimH 100 100 100 93.3 100 88.9

uge 100 100 100 100 100 100

wabG 100 100 100 100 100 100

ureA 100 100 100 100 100 100

(a) The number of tested strains is given in parentheses after ‘n = ’. Values are % of strains with positive PCR reaction.(b) Only K1 strains of CC23K1 are considered.doi:10.1371/journal.pone.0004982.t002



Figure 3. Biotype profiling of K. pneumoniae clones. Cluster analysis (simple matching coefficient) of K. pneumoniae isolates and referencestrains based on metabolic profiles as assessed by biotype-100 strips. Codes above the column correspond to substrate code (Table S3). A bluesquare means the strain grew on the corresponding substrate as sole carbon source. Dark blue, growth was observed after two days; light blue,growth observed after four days. Note the strong homogeneity of biotype-100 profiles within clones. Three clones (Ozaenae, Rhinoscleromatis andCC82 K1) have lost the ability to utilize a number of substrates, including some common substrates between the three clones (see text). Note thatthree tests measure coloration, not growth: hydroxyquinoline-beta-glururonide (black color), tryptophane (brown color: hydrolysis into indole-



onate (0% vs. 100%) and protocatechuate, an intermediate in the

degradation of lignin. Isolates of CC82K1 also had a clearly

distinctive pattern, in particular with the loss of L-fucose, D-

(+)malate and succinate utilization. Clone Ozaenae isolatesexhibited three groups of metabolic profiles (A, B and C on

Figure 3), each of these consisting of the loss of a number ofsubstrates, with trans-aconitate in common. Finally, the remaining

isolates formed a large group that comprised K1 isolates of

CC23K1, showing that these can be differentiated from CC82K1

by the utilization of several carbon sources (Table 3). In addition,CC23K1 were almost exclusive among K. pneumoniae isolates in

using dulcitol and D-tagatose as sole carbon source, while they

differed from the remainder of the large cluster by the loss of

benzoate utilization. Differently, CC14K2 and CC65K2 both

belonged to the large biotype cluster and were weakly distin-

guished, although L-sorbose utilization was found only in CC65.

On average, isolates of the largest cluster were able to utilize

more carbon sources (6563), whereas isolates of clone Rhino-

scleromatis were those with the lowest metabolic abilities (4762.4)(Table 3). CC82K1 and Ozaenae isolates (groups A, B and Ctogether) used 4862.2 and 5065.9 substrates, respectively. It wasstriking that several substrates were lost in common by the three

metabolically-restricted clones. For example, D-Malate and

succinate were lost by Ozaenae (group A) and CC82K1, trans-

aconitate was lost by Ozaenae groups A and B and by CC82K1, 1-

O-Methyl-a-D-glucoside and lactulose were lost by CC82K1 and

Rhinoscleromatis, whereas several substrates (e.g. 5-aminovale-

rate) were lost by the three groups. The loss of the same metabolic

abilities indicates convergent evolution in these clones, possibly

indicative of parallel specialization to a similar niche.

Discussion

The population of K. pneumoniae appears to be characterized by a

low level of nucleotide divergence among orthologous genes,

contrasting with related species such as S. enterica and E. coli. This

pyruvic acid) and histidine (red color). The following substrates were utilized by all assayed strains: D-Glucose, D-fructose, D-trehalose, D-Melibiose, D-Raffinose, Maltotriose, Maltose, D-Cellobiose, 1-O-Methyl-B-D-glucoside, D-Arabitol, Glycerol, Adonitol, N-Acetyl-D-glucosamine, D-Gluconate, L-Alanine and L-Serine. The following substrates were always negative: hydroxyquinoline-beta-glucuronide, D-Lyxose, i-Erythritol, 3-O-Methyl-D-glucose, Tricarballylate, Tryptophan, Gentisate, 3-Hydroxybenzoate, 3-Phenylpropionate, Trigonelline, Betaine, Caprylate, Tryptamine, Itaconate,Propionate, 2-Ketoglutarate.doi:10.1371/journal.pone.0004982.g003

Table 3. Utilization of carbon sources by Klebsiella pneumoniae clones (a).

Substrate code Clone

Rhinoscleromatis Ozaenae CC82K1 CC23K1 CC14K2 CC65K2 Other STs

No. strains: 9 12 9 10 21 8 26

Mean6SD of No. of positive substrates per strain: 4762.4 5065.9 4862.2 6661.4 6561,8 6561.8 6563,4

Carbon sources that discriminate among clones:

D-glucuronate GRT 0 100 100 100 100 100 100

D-galacturonate GAT 0 100 100 100 100 100 100

Palatinose PLE 0 67 100 100 100 100 100

Protocatechuate PAT 0 84 89 100 85 100 100

p-Hydroxybenzoate (4-Hydroxybenzoate) pOBE 0 67 67 100 85 88 95

Mucate MUC 0 67 78 100 100 88 100

trans-Aconitate TATE 89 0 56 100 92 88 95

D(2) Ribose RIB 100 100 0 100 100 100 100

a2L(2) Fucose FUC 100 84 0 67 100 100 95

D(+) Malate DMLT 100 50 11 67 100 100 95

Succinate SUC 100 50 11 100 100 100 95

(2) Quinate QAT 0 50 0 100 85 100 86

Maltitol MTL 0 50 11 100 100 100 100

1-0-Methyl-a2D-glucopyranoside MDG 0 50 0 100 100 75 81

m-Coumarate CMT 0 50 0 100 39 100 86

Lactulose LTE 0 33 0 100 100 100 95

L(+) Sorbose SBE 11 50 0 0 0 88 29

1-0-Methyl-b-galactopyranoside MbGa 11 84 0 100 100 100 100

DL-a-Amino-n-valerate( = 5-Aminovalerate) AVT 0 0 0 100 62 50 62

DL-b-Hydroxybutyrate ( = 3-Hydroxybutyrate) 3OBU 22 0 0 100 100 63 81

Putrescine ( = Diaminobutane) PCE 11 0 0 100 100 100 86

D-Tagatose TAG 0 0 0 100 0 25 43

(a) % positive reactions at day 2.doi:10.1371/journal.pone.0004982.t003



restricted polymorphism cannot be attributed to a biased

sampling, as our dataset included isolates from the environment

and animals, in addition to human isolates from different clinical

sources and large geographic and temporal scales. The genetic

distance that separates K. pneumoniae from its closest phylogeneticrelatives (KpII and KpIV [40,57]) calculated based on the same

seven genes is nearly 4%. Therefore, the species K. pneumoniae may

have undergone a bottleneck relatively recently, long after its

separation from its closest relatives. Still, K. pneumoniae is much

more diverse than notorious monomorphic pathogens such as Y.pestis or S. enterica serotype Typhi [58,59].

A high number of distinct genotypes were disclosed by MLST

despite restricted nucleotide polymorphism. Our analyses suggest

that homologous recombination has more impact on sequence

evolution than mutation, although it is difficult to obtain a reliable

estimate of the recombination/mutation ratio with such a low level

of polymorphism. A high recombination rate would shuffle

polymorphisms among clones and lineages, generating many

genotypes that can be distinguished by MLST. As a consequence,

the clonal frame of K. pneumoniae clones will diversify more rapidlythan it would by a purely mutational process, and the disclosed

STs may not be highly stable over long periods of time.

Gene gnd was atypical by its high level of polymorphism.

Because this gene is located between the rfb and cps operonsresponsible for the synthesis of the two major surface polysaccha-

rides, the lipopolysaccharide and capsule, its evolution is probably

highly influenced by the likely positive selection operating at these

two neighboring loci, as demonstrated for E. coli or Salmonella

[60,61]. In addition, exchange of the cps operon between E. coliand K. pneumoniae was reported [60], and the divergent gnd alleles

encountered in the present study clearly indicate incorporation

into K. pneumoniae isolates of nucleotide sequences from other

Enterobacteriaceae species.

Determining the phylogenetic relationships within a recombin-

ing species is difficult and may even be meaningless if

recombination has erased the pattern of descent among strains.

In particular, analysis based on allelic profiles can be misleading

and may result in the clustering of unrelated STs into long straggly

chains of genotypes [62]. One can therefore be suspicious about

the true clonal link between isolates of CC17, which consists of

chains of STs with distinct K-types, with the exception of some

possibly meaningful terminal groupings such as three K5 STs

(Figure 1).

Identification of clones within species with high rates of

recombination is possible if these clones spread in the population

[63,64]. The fact that several clonal complexes disclosed herein

are relatively homogeneous with respect to several features

including K type, virulence factor content and metabolic profile,

demonstrates that they correspond to clones, i.e. descend from a

common ancestral strain from which they inherited their common

properties. So far, K serotyping has been the dominant common

language for recognition of related Klebsiella strains in epidemio-logical and virulence studies, but it was unknown whether isolates

with the same K type belonged to single clones. Our data clearly

reject this simple view. Indeed, most K-types (with the exception of

K4) that were represented by several isolates were dispatched in

unrelated STs. We could show that the shared K type resulted

from horizontal transfer of the cps operon among these unrelatedgenotypes, generally with the co-transfer of the adjacent gnd gene.Therefore, knowledge of the K type provides unreliable prediction

of clone identity. Given their close physical linkage, recombination

between gnd and cps is probably unfrequent, and gnd sequencing

could therefore be used as a proxy for K typing, which is

technically demanding [50,65,66]. However, the finding of

unrelated gnd sequences in the two major CCs of K2 isolatesshows that this method would not be totally reliable.

Isolates with serotypes K1 to K4 were preferentially included in

this study; therefore, our isolate collection does not reflect K type

frequency in natural populations. Our selected collection allowed

the discovery of six clones comprising isolates that are considered

as particularly pathogenic based on clinical features in animal and

humans and on experimental evidence [1,32–35]. Our data

provide the first evidence that the agent of rhinoscleroma on the

one hand, and isolates recovered from cases of ozaena on the other

hand, each correspond to a single clone. It is remarkable that these

highly homogeneous clones include isolates that were isolated over

a time span of several decades from several countries in Asia,

Africa and Europe. Hence, these two pathogens, both involved in

chronic infections, can be viewed as monomorphic pathogens,

similar in this respect to e.g. Mycobacterium leprae [67]. Nevertheless,

isolates of clone Ozaenae appear to be slightly more heteroge-

neous than Rhinoscleromatis based on MLST data, K type and

biotype. Ozaenae isolates have also been implicated in distinct

types of infections such as bacteremia, urinary tract infections [68]

or splenic abscess [69], and were variable for the presence of

several virulence factors. These observations may reflect a more

diverse lifestyle for Ozaenae than for the intracellular human-

restricted pathogen Rhinoscleromatis.

‘‘K. ozaenae’’ and ‘‘K. rhinoscleromatis’’ could not be separated from

K. pneumoniae by DNA relatedness [70]. For this reason, K. ozaenaeand K. rhinoscleromatis were treated as subspecies of K. pneumoniae in

the early editions of the Bergey’s Manual [18,71]. However, these

two clones appear to have evolved from the genetic pool

taxonomically regarded as K. pneumoniae subsp. pneumoniae, which

does not form a phylogenetic lineage distinct from the other two

subspecies (this study and [40]). Therefore, it is appropriate to

consider isolates associated to rhinoscleroma and ozaena as clones

of K. pneumoniae that acquired particular pathogenic properties,

rather than separate phylogenetic entities that deserve subspecies

status. Our data do not indicate a close affiliation of clone

Rhinoscleromatis with clone Ozaenae. The uncultivable agent of

donovanosis, or granuloma inguinale, has been included in the

genus Klebsiella as K. granulomatis [26,72]. Its phoE sequence [26],

allele phoE-1, was encountered in several K. p. subsp. pneumoniae STs(including CC14K2), and is distinct from phoE-15 found in

Rhinoscleromatis. Despite the similarities in the pathologies they

cause [21,25,26], it was thus not possible to equate K. granulomatiswith clone Rhinoscleromatis, but a close evolutionary link cannot

be excluded. In any case, phoE data indicate that K. granulomatisdoes not represent a distinct genomic species, but instead belongs

to K. pneumoniae as well.

This study demonstrates for the first time that K1 isolates that

cause PLA are genetically distinct from K1 isolates from cases of

respiratory infections and septicemia. Even though our identifica-

tion of CC23K1 as the only clone associated with PLA is based on

only four PLA-causing isolates, this result is fully consistent with a

previous report based on a worldwide collection [38]. Recent

progress stimulated by the emergence of K. pneumoniae PLA hasprovided important clues as to the bacterial factors involved in this

infection [30,31,52,73]. Our PCR tests show that among the

genetic factors that have been associated with K1 PLA strains,

only allS appears to be totally specific for this pathogen. In

addition, we show for the first time that allS is not universallypresent in K1 strains [30,74]. Our data provide the novel

observation that CF29K is particularly prevalent in this clone.

CF29K corresponds to adhesin CS31A found in E. coli strains and

involved in human diarrhea and in septicemia in calves [55]. Our

data suggest that this factor could either be directly implicated in



the pathogenesis of PLA, or linked to another pathogenicity factor

on the 185 kb plasmid that harbors gene cf29A [75]. In agreement

with others [76], we found that magA is present in K1 isolates not

involved in PLA and should thus not be considered as a marker of

PLA-causing isolates [52]. The respiratory or blood origin of most

CC82K1 isolates, together with previous reports of the frequent

implication of K1 strains in Friedländer’s pneumonia, is consistent

with this clone being a prominent agent of this severe form of

pneumonia. The existence of two K1 groups that differ by their

pathological potential is of high relevance for understanding the

bacterial determinants of PLA and acute pneumonia caused by K.

pneumoniae. K2 isolates also clearly appear to be distributed into

several unrelated genotypes. For both K1 and K2 serotypes, we

could show that the pathogenic potential of strains depends on

their genotype, rather than on their K type. CC14K2 comprises

isolates for serotypes K2 and K24, but we did not observe any

difference in virulence gene content or in virulence to mice

between CC14K2 members of both serotypes. In contrast, some

virulence factors distinguished CC14K2 (including its K24 isolates)

and CC65K2, e.g. gene kfu (100% vs. 0%, respectively). These

results show that at least for K1 and K2 isolates, the clonal

complex is a better predictor of virulence gene content and of

virulence to mice than the K type, and that previous associations

of virulence factors with K-types [74,77] should be revisited by

analysis of isolates from distinct CCs. Thus, even if the capsular

polysaccharide is a prominent pathogenicity determinant, the

long-held belief that K type is predictive of virulence should be

discontinued.

The nature of K. pneumoniae pathogenic clones and their history

of interaction with their animal and human hosts, including

possible specialization, remain largely unknown. Our biotype data

demonstrated that the three clones Rhinoscleromatis, Ozaenae

and CC82K1 have each lost several metabolic abilities, some of

which in common, probably by parallel evolution. It has long been

recognized that the two former clones are biotypes of K. pneumoniae

with less nutritional versatility [18,27] and together with some K1

strains, require specific factors for growth [27]. To our knowledge,

Rhinoscleromatis, Ozaenae and K1 isolates have neither been

reported from the environment, nor in intestinal carriage, and it is

perhaps significant that several substrates that are not utilized by

these clones belong to plant product degradation pathways. We

hypothesize that these three clones are engaged in evolutionary

specialization to a restricted ecological niche, possibly represented

by the upper respiratory tract of humans. A restriction in

ecological niche may in turn reduce the opportunity for encounter

with other K. pneumoniae strains. The intracellular lifestyle ofRhinoscleromatis provides the most achieved example, and this

pathogen may now be evolving independently from its ancestral

species K. pneumoniae. Consistent with this hypothesis, Rhinoscler-

omatis and Ozaenae were genetically the most distinct of the 117

STs (Figure 1), which had in general no more than three allelicmismatches among themselves. This observation suggests that

these two clones are less frequently involved in allelic exchange

with other strains. Finally, it is interesting to notice that the gene

coding for the adhesin MrkD was undetected specifically in

CC82K1 and Ozaenae, also suggestive of niche reduction. In

contrast, the typical biotype profile of the PLA-associated CC23K1

does not suggest ecological specialization. Hence, the acquisition

by this clone of its particular set of virulence determinants is

possibly recent in time, consistent with epidemiological data [12],

and the pathogenicity of clone CC23K1 may be uncoupled from

any particular adaptation to humans. Infection of the liver is

believed to take place from the intestine. Because liver infection

and metastasis to the eye and brain are unlikely to provide any

specific selective advantage to this clone, pathogenesis can be

viewed as accidental. Given that the natural habitat of this clone is

probably indistinct from its non-virulent ancestor, keeping an

intact metabolic versatility may be a key requirement for successful

competition of this clone with other generalist K. pneumoniae clones.

It is therefore unlikely that reductive evolution by specialization

will be observed in this important emerging clone.

Materials and Methods

Bacterial isolatesA total of 235 K. pneumoniae reference strains or isolates were

included in this study (Table S1). Capsular (K) serotypes K1 toK4 were included preferentially in order to estimate their genetic

diversity. The collection included 25 isolates with serotype K1

from cases of pyogenic liver abscess (n = 4), other clinical sources

(n = 17) and reference strains (n = 4). Nineteen K2 isolates, 16 K.pneumoniae subsp. rhinoscleromatis isolates (all being K3) and 14 K.pneumoniae subsp. ozaenae (12 K4 and 2 K5) were included. Forcomparison purposes, we included K3 (n = 10) and K5 isolates

(n = 4) of K. pneumoniae subsp. pneumoniae. Type strains of the threesubspecies and reference strains of serotypes K1 to K5 as well as

laboratory strain KP52.145, were included. In some cases (TableS1, column ‘probable duplicate’), two or more subcultures of thesame original strain were included, because they were obtained

from different sources (e.g., the Orskov collection of K-type

reference strains, the Collection de l’Institut Pasteur [CIP] and the

ATCC). This is due to the fact that the K-type reference strain and

the taxonomic type strain or other laboratory strains are

sometimes derived from the same initial strain.

The remaining isolates were included to represent different

sources, without consideration of their K type. Most isolates were

of human clinical origin. For comparison purposes, we collected

13 isolates from the environment and 18 from fecal samples using

a selective medium based on citrate and inositol [78], and

gathered 30 horse isolates and 8 other animal isolates from

previous studies [49,79–81]. The 67 isolates from nosocomial

infections previously analyzed [82] were included. Isolates

originated from 20 countries from Europe, North America, Asia

and Africa. The most represented countries were France and the

Netherlands (Table S1).

Species and subspecies identification and biotypingIsolates were initially identified as Klebsiella pneumoniae sensu stricto,

K. pneumoniae subsp. rhinoscleromatis or K. pneumoniae subsp. ozaenaeusing standard, recommended biochemical tests [27]. Identifica-

tion of the later two subspecies was controlled by capsular

serotyping using the capsular swelling method and using the

following biochemical tests: Voges-Proskauer, urease, ONPG,

lysine decarboxylase, citrate, malonate, and gas production.

Isolates could be identified as belonging to K. pneumoniae sensustricto, i.e., phylogenetic group KpI [40], based on phylogeneticclustering of the seven MLST genes and gyrA [40], using KpII-A,KpII-B [57], KpIII [40] and K. oxytoca, K. planticola and K. terrigenaisolates for comparison.

Re-identification and biotyping of isolates at the species level (or

subspecies level within K. pneumoniae) was performed using Biotype-100 strips (BioMérieux, Marcy l’Etoile, France), which contain 99

substrates in cupules [27]. Minimal medium 1 was used and

isolates were identified using software Recognizer (P.A.D.

Grimont, Institut Pasteur) against the Enterobacteriaceae databaseconstructed in the laboratory (version 2000). Substrates that were

particularly useful for species discrimination were m-coumarate,

gentisate, histamin, 3-hydroxybenzoate, D-melezitose, 3-O-meth-



yl-D-glucose, and tricarballylate [27]. Minimal medium 2 was

used for isolates of K. pneumoniae subsp. rhinoscleromatis or K.

pneumoniae subsp. ozaenae [27]. Reproducibility of biotype-100profiles was controlled by inclusion of strain ATCC 13883T in

each batch and by the independent analysis of synonymous strains

(Table S1; Figure 3).

Capsular serotypingSerotyping was determined by the capsular swelling method

[49,79,80], and the K-type of some isolates were controlled by the

agglutination method [65]. The K-serotype of the type strains and

reference strains was known prior to this study.

cps PCR-RFLP (molecular serotyping)The determination of the C-pattern was determined as

previously described [50]. A reference C-pattern database was

constituted by the C-patterns obtained for the 77 reference strains

of the International serotyping scheme and for the study isolates

for which the K-type was determined by classical serotyping. C-

patterns that were encountered in isolates of defined capsular type

were labeled with ‘C’ followed by the number capsular (e.g., C2)

followed by a letter denoting the successive banding patterns found

for isolates of this serotype (e.g., C2a, C2b …). Some isolates were

analyzed by cps PCR-RFLP but not by classical serotyping. For theC-patterns that had a match in the reference database, the same

K-type as that of the reference strain(s) was inferred. For the

remaining isolates, the obtained C-pattern had no match in the

reference database; these C-patterns were numbered consecutive-

ly, starting at C100 (Table S1).

Multilocus Sequence Typing (MLST)MLST was performed as previously described [82] with the

following modification: universal sequences were added upstream

of each forward (GTT TTC CCA GTC ACG ACG TTG TA)

and reverse (TTG TGA GCG GAT AAC AAT TTC) primers. All

amplifications were performed at 50uC, and sequences wereobtained using the two universal sequencing primers given above.

Further details are available on the K. pneumoniae MLST web site

(www.pasteur.fr/mlst).

gnd gene sequencingThe sequence of a 360-bp portion of the gnd gene was

established on both strands by using primers gnd-1F (TGA

AGC AGC AAA CAA AGG TAC) and gnd-8R (TCA TCG

GCG ATC TGC TTA AAG T), which amplify an internal

portion of 457 bp of the gene. The annealing temperature was

46uC (30 cycles of 30 sec, 94uC; 30 sec., 46uC; 30 sec., 72uC,followed by 1 min at 72uC). When amplification failed, primergnd-2 (ACA TCA CGC AGC GCC TGC TGA T) was used

instead of gnd-8R, with 50uC as annealing temperature.Sequencing primers were gnd-9, TGA TGA (A/G)GC nGC (A/

c)AA CAA AGG TAC, and gnd-10, TCA TCa GC(a/G) ATC

TG(C/t) TTG AAG Ta(c/t).

Virulence PCRPCR assays were performed to check for the presence of 10

genes that have previously been associated with virulence in K.

pneumoniae. Target genes, primers used and specific annealing

temperature of PCR are given in Table S2. After 5 min at 94uC,there were 35 cycles of 94uC, 30 sec.; annealing temperature,30 sec.; and 72uC, 1 min. followed by a final elongation of 1 minat 72uC. Strains NTUH-K2044, KP52145 and MGH 78578[31,34,52,83] were used as PCR controls. PCR products from

several STs were systematically sequenced to control that the

amplified PCR products corresponded to the expected gene.

Infection of miceFemale Balb/cJ mice were purchased from R. Janvier Breeding

Center (Le Genest St. Isle, France) and housed under standard

conditions of feeding, light and temperature with free access to

food and water. Experiments were performed according to the

Institut Pasteur guidelines for laboratory animals husbandry.

Seven to eight weeks-old mice were first anesthetized, with 80

microliters intramuscular injection of ketamine (Imalgene,

31.25 mg/kg, Merial) and Acepromazine (Calmivet, 1.5 mg/kg,

Vetoquinol) and then infected by inoculation of 20 microliters of

bacteria suspension (106 bacteria) into their right nostril. Eight

mice per strain were infected. The number of surviving mice was

monitored every day during twelve days.

Data analysisFor each MLST locus, an allele number was given to each

distinct sequence variant (confirmed by at least two chromatogram

traces), and a distinct sequence type (ST) number was attributed to

each distinct combination of alleles at the seven genes. Allele and

profile numbers were incremented successively in the order in

which they were discovered. In order to define the relationships

among isolates at the microevolutionary level, we performed allelic

profile – based comparisons using a minimum spanning tree

(MStree) analysis with the BioNumerics v5.10 software (Applied-

Maths, Sint Maartens-Latem, Belgium). MStree analysis links

profiles so that the sum of the distances (number of distinct alleles

between two STs) is minimized [84]. Isolates were grouped into

clonal complexes (clonal families), defined as groups of profiles

differing by no more than one gene from at least one other profile

of the group [85]. Accordingly, singletons were defined as STs

having at least two allelic mismatches with all other STs.

Split decomposition analysis was performed using SplitsTree

version 4.10 [86,87]. Neighbor-joining tree analysis was per-

formed using MEGA v4 [88]. Nucleotide diversity indices were

calculated using DNAsp v4 [89]. ClonalFrame analysis [46] was

performed with 50,000 burn-in iterations and 100,000 subsequent

iterations.

The relative contribution of recombination and mutation on the

short term was calculated using eBURST and the clonal

diversification method [90,91]. For each pair of allelic profiles

that differ by a single allelic mismatch (single locus variants, or

SLVs), the number of nucleotide changes between the alleles that

differ is counted. A single nucleotide difference is considered to be

likely caused by mutation, whereas more than one mutation in the

same gene portion is considered to derive from recombination, as

it is considered unlikely that two mutations would occur on the

same gene while the other genes remain identical. No correction

was made for single nucleotide differences possibly introduced by

recombination.

The population recombination rate was estimated by a

composite-likelihood method with LDHAT [92]. LDHAT employs

a parametric approach, based on the neutral coalescent, to

estimate the scaled parameter 2Ner where Ne is the effectivepopulation size, and r is the rate at which recombination eventsseparate adjacent nucleotides. The crossing-over model L was used

for the analysis of biallelic sites, with frequency of the less frequent

allele .0.1.

Nucleotide sequencesSequences generated in this study are available at www.pasteur.

fr/mlst for the seven MLST genes. In addition, gnd alleles have



been deposited in GenBank/EMBL/DDBJ databases under the

accession numbers FJ769917-FJ769969.

Supporting Information

Table S1 Strains. Characteristics of the 235 strains included in

the study.

Found at: doi:10.1371/journal.pone.0004982.s001 (0.13 MB

XLS)

Table S2 Primers used for virulence genes PCR.


XLS)

Table S3 Carbon sources assayed with the Biotype-100 strips.


PDF)

Acknowledgments

We are grateful to Engeline van Duijkeren, J. Verhoef, A. Fluit, Jin-Town

Wang, Etienne Carbonelle and Christophe De Champs for providing

isolates. The Collection de l’Institut Pasteur is acknowledged for providing

reference and type strains. Our study benefited greatly from the historical

Klebsiella collection of the Institut Pasteur Enterobacteriaceae Unit, gathered by

the late Claude Richard.

Author Contributions

Conceived and designed the experiments: SB CF RT PG. Performed the

experiments: SB CF VP SIJ RT LD. Analyzed the data: SB CF RT. Wrote

the paper: SB.

References

1. Ørskov I (1981) The genus Klebsiella (Medical aspects). In: Starr MP, Stolp H,Truper HG, Balows A, Schlegel HG, eds. The Prokaryotes: a handbook on

habitats, isolation, and identification of bacteria. Berlin: Springer-Verlag. pp

1160–1165.

2. Seidler RJ (1981) The genus Klebsiella (Nonmedical aspects). In: Starr MP,Stolp H, Truper HG, Balows A, Schlegel HG, eds. The Prokaryotes: a

handbook on habitats, isolation, and identification of bacteria. Berlin: Springer-

Verlag. pp 1166–1172.

3. Brisse S, Grimont F, Grimont PAD (2006) The genus Klebsiella. In: Dworkin M,Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, eds. The Prokaryotes ?A Handbook on the Biology of Bacteria. 3rd edition ed. New York: Springer.

4. Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens:epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin

Microbiol Rev 11: 589–603.

5. Paterson DL, Ko WC, Von Gottberg A, Mohapatra S, Casellas JM, et al. (2004)

International prospective study of Klebsiella pneumoniae bacteremia: implications ofextended-spectrum beta-lactamase production in nosocomial Infections. Ann

Intern Med 140: 26–32.

6. Woodford N, Reddy S, Fagan EJ, Hill RL, Hopkins KL, et al. (2007) Wide

geographic spread of diverse acquired AmpC beta-lactamases among Escherichiacoli and Klebsiella spp. in the UK and Ireland. J Antimicrob Chemother 59:102–105.

7. Colodner R, Rock W, Chazan B, Keller N, Guy N, et al. (2004) Risk factors for

the development of extended-spectrum beta-lactamase-producing bacteria in

nonhospitalized patients. Eur J Clin Microbiol Infect Dis 23: 163–167.

8. Keynan Y, Rubinstein E (2007) The changing face of Klebsiella pneumoniaeinfections in the community. Int J Antimicrob Agents 30: 385–389.

9. Carpenter JL (1990) Klebsiella pulmonary infections: occurrence at one medicalcenter and review. Rev Infect Dis 12: 672–682.

10. Ko WC, Paterson DL, Sagnimeni AJ, Hansen DS, Von Gottberg A, et al. (2002)

Community-acquired Klebsiella pneumoniae bacteremia: global differences inclinical patterns. Emerg Infect Dis 8: 160–166.

11. Yu VL, Hansen DS, Ko WC, Sagnimeni A, Klugman KP, et al. (2007)

Virulence characteristics of Klebsiella and clinical manifestations of K. pneumoniaebloodstream infections. Emerg Infect Dis 13: 986–993.

12. Liu YC, Cheng DL, Lin CL (1986) Klebsiella pneumoniae liver abscess associatedwith septic endophthalmitis. Arch Intern Med 146: 1913–1916.

13. Cheng DL, Liu YC, Yen MY, Liu CY, Wang RS (1991) Septic metastatic lesions

of pyogenic liver abscess. Their association with Klebsiella pneumoniae bacteremiain diabetic patients. Arch Intern Med 151: 1557–1559.

14. Wang JH, Liu YC, Lee SS, Yen MY, Chen YS, et al. (1998) Primary liver

abscess due to Klebsiella pneumoniae in Taiwan. Clin Infect Dis 26: 1434–1438.

15. Fang FC, Sandler N, Libby SJ (2005) Liver abscess caused by magA+ Klebsiellapneumoniae in North America. J Clin Microbiol 43: 991–992.

16. Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, et al. (2007) Klebsiella pneumoniaegenotype K1: an emerging pathogen that causes septic ocular or central nervous

system complications from pyogenic liver abscess. Clin Infect Dis 45: 284–293.

17. Rahimian J, Wilson T, Oram V, Holzman RS (2004) Pyogenic liver abscess:

recent trends in etiology and mortality. Clin Infect Dis 39: 1654–1659.

18. Ørskov I (1984) Klebsiella Trevisan 1885. In: Krieg NR, Holt JG, eds. Bergey’smanual of systematic bacteriology. Baltimore: Williams and Wilkins Co. pp

461–465.

19. Chand MS, MacArthur CJ (1997) Primary atrophic rhinitis: a summary of four

cases and review of the litterature. Otolaryngol Head Neck Surg 116: 554–558.

20. Shehata MA (1996) Atrophic rhinitis. Am J Otolaryngol 17: 81–86.

21. Hart CA, Rao SK (2000) Rhinoscleroma. J Med Microbiol 49: 395–396.

22. Lu CH, Chang WN, Chang HW (2002) Klebsiella meningitis in adults: clinicalfeatures, prognostic factors and therapeutic outcomes. J Clin Neurosci 9:

533–538.

23. Wong CH, Kurup A, Wang YS, Heng KS, Tan KC (2004) Four cases of

necrotizing fasciitis caused by Klebsiella species. Eur J Clin Microbiol Infect Dis23: 403–407.

24. Kohler JE, Hutchens MP, Sadow PM, Modi BP, Tavakkolizadeh A, et al. (2007)

Klebsiella pneumoniae necrotizing fasciitis and septic arthritis: an appearance in theWestern hemisphere. Surg Infect (Larchmt) 8: 227–232.

25. Richens J (1985) Donovanosis–a review. P N G Med J 28: 67–74.

26. Carter JS, Bowden FJ, Bastian I, Myers GM, Sriprakash KS, et al. (1999)

Phylogenetic evidence for reclassification of Calymmatobacterium granulomatis asKlebsiella granulomatis comb. nov. Int J Syst Bacteriol 49: 1695–1700.

27. Grimont PAD, Grimont F (2005) Genus Klebsiella. In: Brenner DJ, Krieg NR,Staley JT, eds. Bergey’s manual of Systematic Bacteriology Volume 2: The

Proteobacteria, Part B: The Gammaproteobacteria. New York: Springer-Verlag.

pp 685–693.

28. Williams P, Tomas JM (1990) The pathogenicity of Klebsiella pneumoniae. Reviewsof Medical Microbiology 1: 196–204.

29. Regue M, Climent N, Abitiu N, Coderch N, Merino S, et al. (2001) Genetic

characterization of the Klebsiella pneumoniae waa gene cluster, involved in corelipopolysaccharide biosynthesis. J Bacteriol 183: 3564–3573.

30. Chou HC, Lee CZ, Ma LC, Fang CT, Chang SC, et al. (2004) Isolation of a

chromosomal region of Klebsiella pneumoniae associated with allantoin metabolismand liver infection. Infect Immun 72: 3783–3792.

31. Ma LC, Fang CT, Lee CZ, Shun CT, Wang JT (2005) Genomic heterogeneity

in Klebsiella pneumoniae strains is associated with primary pyogenic liver abscessand metastatic infection. J Infect Dis 192: 117–128.

32. Simoons-Smit AM, Verweiji-van Vught AMJJ, Kanis IYR, MacLaren DM

(1984) Virulence of Klebsiella strains in experimentally induced skin lesions in themouse. Journal of Medical Microbiology 17: 67–77.

33. Mizuta LG, Ohta M, Mori M, Hasegawa T, Nakashima I, Kato N (1983)

Virulence for mice of Klebsiella strains belonging to the O1 group: relationship totheir capsular (K) types. Infect Immun 40: 56–61.

34. Nassif X, Sansonetti PJ (1986) Correlation of the virulence of Klebsiella pneumoniaeK1 and K2 with the presence of a plasmid encoding aerobactin. Infection and

Immunity 54: 603–608.

35. Ofek I, Kabha K, Athamna A, Frankel G, Wozniak DJ, et al. (1993) Genetic

exchange of determinants for capsular polysaccharide biosynthesis between

Klebsiella pneumoniae strains expressing serotypes K2 and K21a. Infect Immun 61:4208–4216.

36. Eickoff TC, Steinhauer BW, Finland M (1966) The Klebsiella-Enterobacter-Serratiadivision. Biochemical and serological characteristitcs and susceptibility to

antibiotics. Annales of Internal Medicine 65: 1163–1179.

37. Cheng HP, Chang FY, Fung CP, Siu LK (2002) Klebsiella pneumoniae liver abscessin Taiwan is not caused by a clonal spread strain. J Microbiol Immunol Infect

35: 85–88.

38. Turton JF, Englender H, Gabriel SN, Turton SE, Kaufmann ME, et al. (2007)

Genetically similar isolates of Klebsiella pneumoniae serotype K1 causing liverabscesses in three continents. J Med Microbiol 56: 593–597.

39. Coffey TJ, Dowson CG, Daniels M, Zhou J, Martin C, et al. (1991) Horizontal

transfer of multiple penicillin-binding protein genes, and capsular biosynthetic

genes, in natural populations of Streptococcus pneumoniae. Mol Microbiol 5:2255–2260.

40. Brisse S, Verhoef J (2001) Phylogenetic diversity of Klebsiella pneumoniae andKlebsiella oxytoca clinical isolates revealed by randomly amplified polymorphicDNA, gyrA and parC genes sequencing and automated ribotyping. Int J Syst EvolMicrobiol 51: 915–924.

41. Nouvellon M, Pons JL, Sirot D, Combe ML, Lemeland JF (1994) Clonal

outbreaks of extended-spectrum beta-lactamase-producing strains of Klebsiellapneumoniae demonstrated by antibiotic susceptibility testing, beta-lactamasetyping, and multilocus enzyme electrophoresis. J Clin Microbiol 32: 2625–2627.



42. Maurelli AT, Fernandez RE, Bloch CA, Rode CK, Fasano A (1998) ‘‘Black

holes’’ and bacterial pathogenicity: a large genomic deletion that enhances thevirulence of Shigella spp. and enteroinvasive Escherichia coli. Proc Natl AcadSci U S A 95: 3943–3948.

43. Dagan T, Blekhman R, Graur D (2006) The ‘‘domino theory’’ of gene death:

gradual and mass gene extinction events in three lineages of obligate symbiotic

bacterial pathogens. Mol Biol Evol 23: 310–316.

44. Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, et al. (2001)

Complete genome sequence of a multiple drug resistant Salmonella enterica serovarTyphi CT18. Nature 413: 848–852.

45. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimatelarge phylogenies by maximum likelihood. Syst Biol 52: 696–704.

46. Didelot X, Falush D (2007) Inference of bacterial microevolution usingmultilocus sequence data. Genetics 175: 1251–1266.

47. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG (2004) eBURST:inferring patterns of evolutionary descent among clusters of related bacterial

genotypes from multilocus sequence typing data. J Bacteriol 186: 1518–1530.

48. Van Westreenen M, Paauw A, Fluit AC, Brisse S, Van Dijk W, et al. (2003)

Occurrence and spread of SHV extended-spectrum beta-lactamase-producingKlebsiella pneumoniae isolates in Curaçao. J Antimicrob Chemother 52: 530–532.

49. Richard C (1989) Epidémiologie des infections à Klebsiella pneumoniae dans deuxélevages de singes-écureuils et de lémuriens. Bull Soc Pathol Exot 82: 458–464.

50. Brisse S, Issenhuth-Jeanjean S, Grimont PA (2004) Molecular serotyping of

Klebsiella species isolates by restriction of the amplified capsular antigen genecluster. J Clin Microbiol 42: 3388–3398.

51. Arakawa Y, Wacharotayankun R, Nagatsuka T, Ito H, Kato N, et al. (1995)Genomic organization of the Klebsiella pneumoniae cps region responsible forserotype K2 capsular polysaccharide synthesis in the virulent strain Chedid.J Bacteriol 177: 1788–1796.

52. Fang CT, Chuang YP, Shun CT, Chang SC, Wang JT (2004) A novel virulencegene in Klebsiella pneumoniae strains causing primary liver abscess and septicmetastatic complications. J Exp Med 199: 697–705.

53. Hornick DB, Allen BL, Horn MA, Clegg S (1992) Adherence to respiratory

epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3fimbrial gene products. Infect Immun 60: 1577–1588.

54. Tarkkanen AM, Virkola R, Clegg S, Korhonen TK (1997) Binding of the type 3

fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells.Infect Immun 65: 1546–1549.

55. Di Martino P, Bertin Y, Girardeau JP, Livrelli V, Joly B, et al. (1995) Molecularcharacterization and adhesive properties of CF29K, an adhesin of Klebsiellapneumoniae strains involved in nosocomial infections. Infect Immun 63:4336–4344.

56. Nassif X, Fournier J-M, Arondel J, Sansonetti PJ (1989) Mucoid phenotype ofKlebsiella pneumoniae is a plasmid-encoded virulence factor. Infection andImmunity 57: 546–552.

57. Fevre C, Passet V, Weill FX, Grimont PA, Brisse S (2005) Variants of the

Klebsiella pneumoniae OKP chromosomal beta-lactamase are divided into twomain groups, OKP-A and OKP-B. Antimicrob Agents Chemother 49:

5149–5152.

58. Roumagnac P, Weill FX, Dolecek C, Baker S, Brisse S, et al. (2006)Evolutionary history of Salmonella typhi. Science 314: 1301–1304.

59. Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A, et al. (1999) Yersiniapestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis.Proc Natl Acad Sci U S A 96: 14043–14048.

60. Nelson K, Selander RK (1994) Intergeneric transfer and recombination of the 6-

phosphogluconate dehydrogenase gene (gnd) in enteric bacteria. Proc Natl AcadSci U S A 91: 10227–10231.

61. Bisercic M, Feutrier JY, Reeves PR (1991) Nucleotide sequences of the gnd genesfrom nine natural isolates of Escherichia coli: evidence of intragenic recombinationas a contributing factor in the evolution of the polymorphic gnd locus. J Bacteriol173: 3894–3900.

62. Turner KM, Hanage WP, Fraser C, Connor TR, Spratt BG (2007) Assessing the

reliability of eBURST using simulated populations with known ancestry. BMCMicrobiol 7: 30.

63. Smith JM, Smith NH, O’Rourke M, Spratt BG (1993) How clonal are bacteria?Proc Natl Acad Sci U S A 90: 4384–4388.

64. Spratt BG, Maiden MC (1999) Bacterial population genetics, evolution andepidemiology. Philos Trans R Soc Lond B Biol Sci 354: 701–710.

65. Ørskov I, Ørskov F (1984) Serotyping of Klebsiella. In: Bergan T, ed. Methods inMicrobiology. London: Academic Press. pp 143–164.

66. Hansen DS, Skov R, Benedi JV, Sperling V, Kolmos HJ (2002) Klebsiella typing:pulsed-field gel electrophoresis (PFGE) in comparison with O:K-serotyping. Clin

Microbiol Infect 8: 397–404.

67. Monot M, Honore N, Garnier T, Araoz R, Coppee JY, et al. (2005) On the

origin of leprosy. Science 308: 1040–1042.68. Goldstein EJC, Lewis RP, Martin WJ, Edelstein PH (1978) Infections caused by

Klebsiella ozaenae: a changing disease spectrum. Journal of Clinical Microbiology8: 413–418.

69. Sarma PS (2007) Klebsiella ozaenae splenic abscess following odontogenic infectionin a girl with sickle cell disease. Int J Infect Dis 11: 86–87.

70. Brenner DJ, Steigerwalt AG, Fanning GR (1972) Differentiation of Enterobacteraerogenes from Klebsiellae by deoxyribonucleic acid reassociation. InternationalJournal of Systematic Bacteriology 22: 193–200.

71. Ørskov I (1974) Klebsiella. In: Buchanan RE, Gibbons NE, eds. Bergey’s manualof determinative bacteriology. 8th ed. Baltimore: Williams & Wilkins. pp321–324.

72. Kharsany AB, Hoosen AA, Kiepiela P, Kirby R, Sturm AW (1999) Phylogeneticanalysis of Calymmatobacterium granulomatis based on 16S rRNA gene sequences.J Med Microbiol 48: 841–847.

73. Lai YC, Lin GT, Yang SL, Chang HY, Peng HL (2003) Identification andcharacterization of KvgAS, a two-component system in Klebsiella pneumoniaeCG43. FEMS Microbiol Lett 218: 121–126.

74. Yu WL, Ko WC, Cheng KC, Lee CC, Lai CC, et al. (2008) Comparison of

prevalence of virulence factors for Klebsiella pneumoniae liver abscesses betweenisolates with capsular K1/K2 and non-K1/K2 serotypes. Diagn MicrobiolInfect Dis.

75. Darfeuille-Michaud A, Jallat C, Aubel D, Sirot D, Rich C, et al. (1992) R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible forhuman nosocomial infections. Infect Immun 60: 44–55.

76. Struve C, Bojer M, Nielsen EM, Hansen DS, Krogfelt KA (2005) Investigation

of the putative virulence gene magA in a worldwide collection of 495 Klebsiellaisolates: magA is restricted to the gene cluster of Klebsiella pneumoniae capsuleserotype K1. J Med Microbiol 54: 1111–1113.

77. Yeh KM, Kurup A, Siu LK, Koh YL, Fung CP, et al. (2007) Capsular serotypeK1 or K2, rather than magA and rmpA, is a major virulence determinant forKlebsiella pneumoniae liver abscess in Singapore and Taiwan. J Clin Microbiol 45:466–471.

78. van Kregten E, Westerdaal NAC, Willers JMN (1984) New, simple medium for

selective recovery of Klebsiella pneumoniae and Klebsiella oxytoca from human feces.Journal of Clinical Microbiology 20: 936–941.

79. Richard C (1973) Etude antigénique et biochimique de 500 souches de Klebsiella.Annales de Biologie Clinique 31: 295–303.

80. Tainturier D, Richard C (1986) Métrites en série pendant la saison de monte

chez des juments de trait breton provoquées par Klebsiella pneumoniae typecapsulaire K2, biotype d. Revue de Médecine Vétérinaire 137: 49–58.

81. Brisse S, Duijkeren E (2005) Identification and antimicrobial susceptibility of 100Klebsiella animal clinical isolates. Vet Microbiol 105: 307–312.

82. Diancourt L, Passet V, Verhoef J, Grimont PAD, Brisse S (2005) Multilocus

sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 43:4178–4182.

83. McClelland M, Florea L, Sanderson K, Clifton SW, Parkhill J, et al. (2000)Comparison of the Escherichia coli K-12 genome with sampled genomes of aKlebsiella pneumoniae and three Salmonella enterica serovars, Typhimurium, Typhiand Paratyphi. Nucleic Acids Res 28: 4974–4986.

84. Schouls LM, van der Heide HG, Vauterin L, Vauterin P, Mooi FR (2004)

Multiple-locus variable-number tandem repeat analysis of Dutch Bordetellapertussis strains reveals rapid genetic changes with clonal expansion during thelate 1990s. J Bacteriol 186: 5496–5505.

85. Feil EJ (2004) Small change: keeping pace with microevolution. Nat Rev

Microbiol 2: 483–495.

86. Huson DH (1998) SplitsTree: analyzing and visualizing evolutionary data.Bioinformatics 14: 68–73.

87. Huson DH, Bryant D (2006) Application of phylogenetic networks inevolutionary studies. Mol Biol Evol 23: 254–267.

88. Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular

evolutionary genetics analysis software. Bioinformatics 17: 1244–1245.89. Rozas J, Rozas R (1999) DnaSP version 3: an integrated program for molecular

population genetics and molecular evolution analysis. Bioinformatics 15:174–175.

90. Feil EJ, Maiden MC, Achtman M, Spratt BG (1999) The relative contributionsof recombination and mutation to the divergence of clones of Neisseria meningitidis.Mol Biol Evol 16: 1496–1502.

91. Guttman DS, Dykhuizen DE (1994) Clonal divergence in Escherichia coli as aresult of recombination, not mutation. Science 266: 1380–1383.

92. McVean G, Awadalla P, Fearnhead P (2002) A coalescent-based method fordetecting and estimating recombination from gene sequences. Genetics 160:

1231–1241.



Virulent Clones of Klebsiella pneumoniae: Identification...

Documents

Transcript of Virulent Clones of Klebsiella pneumoniae: Identification...